An investigation of proteins produced by porcine oviduct epithelial cells cultured in a serum-free medium

The objective of the present study was to establish a serum-free culture system for porcine oviduct epithelial cells, and to investigate the proteins secreted by the cultured epithelial cells. Epithelial cells separated from oviductal stroma by EDTA and collagenase treatment grew successfully in a serum-free culture medium composed of a 1:1 mixture of DMEM: Ham's F12 supplemented with insulin (10 mug/ml), transferrin (10 mug/ml), sodium selenite (25 etag/ml) and antibiotics. The cells grew to a confluent monolayer on the collagen gel within 6 days after seeding. The confluent monolayer state was maintained for at least 2 weeks. The epithelial nature of cultured cells was confirmed by transmission electron microscopic observation of desmosomes and microvilli. A high viability (79.5 %) was obtained in frozen-thawed cells when a serum-free medium containing 10 % DMSO was supplemented with 0.1 % methylcellulose. The addition of 10 -6M 17beta-estradiol (E2) to the culture medium from day 6 to day 8 resulted in the extension of the culture term from 2 weeks to 3 weeks. However, analysis of the conditioned medium derived from cultured cells by SDS-PAGE revealed that no specific bands were observed by an addition of 10 -5 - 10 -10 ME2. Porcine ampullary and isthmic epithelial cells were grown separately in a serum-free culture medium. Different protein bands were observed between the ampulla and isthmus after a SDS-PAGE analysis of the conditioned medium. Cell growth activity in conditioned medium from ampullary and isthmic epithelial cells was assessed by a colony formation method in which one cell proliferates to form a colony. The colony formation rate in an assay medium from the ampullary and isthmic cells was 33 % and 19 %, respectively, while a colony formation rate of only 2 % was observed in the control assay medium. The present study suggests that porcine oviduct synthesize and secrete specific proteins to stimulate cell growth and that a difference in cell growth activity exists between the ampullary and isthmic regions